Veterinary Science - Theses
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ItemAnalysis of physiological death in equine chondrocytes( 2007-07)Chondrocytes in growth cartilage undergo proliferation, hypertrophy, and then die by a mechanism that has not been characterised. The aims of the current study were to document the morphology of dying hypertrophic chondrocytes in equine growth cartilage and to establish a culture system in which the isolated chondrocytes can be induced to undergo the same modes of hypertrophy and physiological death seen in growth cartilage in vivo. Growth cartilage from foetal and growing postnatal horses was examined by electron microscopy. Ultrastructural studies of the tissue specimens suggested that the two types of hypertrophic chondrocytes that have previously been described as dark and light cells were dying by different non-apoptotic forms of cell death. Dying hypertrophic dark chondrocytes were characterised by a dark nucleus, and their cytoplasm appeared to undergo extrusion into the extracellular matrix, whereas light chondrocytes appeared to disintegrate within the cell membrane.
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ItemMolecular pathogenesis of chicken anaemia virus( 2007)Chicken anaemia virus (CAV) causes a disease in young chickens characterised by aplastic anaemia and generalized lymphoid atrophy. CAV has three major overlapping open reading frames (ORF), encoding proteins VP1, VP2 and VP3. VP3 can induce apoptosis in tumour cell lines and the ORF that encodes it is completely encompassed by the ORF encoding VP2. Previous studies at the University of Melbourne used site-directed mutagenesis of VP2 to generate several CAV mutants and the potential of these mutants as CAV vaccines was assessed in subsequent studies. The effect of CAV mutants on the thymus was investigated by histopathological examination and immunofluorescent staining. A scoring system based on specific medullary cells in the thymus failed to distinguish between birds inoculated with wild type CAV and those inoculated with RPMI medium. Thymic sections of birds inoculated with CAV mutants were stained with an anti-CD8 monoclonal antibody and studied by fluorescence microscopy. The thymic cortical thicknesses were measured digitally and analysed using the Image J program. The results showed that the mutants E186G and R/K/K150/151/152G/A/A were attenuated and that mutant S77N was less attenuated. Vaccination with mutants Q131P, E186G and R/K/K150/151/152G/A/A resulted in an increase in the median thymic cortical thickness, suggesting greater repopulation by CD8+ lymphocytes. A SYBR Green quantitative polymerase chain reaction assay was developed to evaluate serum neutalisation (SN) assays on the sera of birds inoculated with the CAV mutants. Mutant E186G induced the highest neutralizing antibody titres, followed by mutants Q131P, S77N and R/K/K150/151/152G/A/A. One-day-old and six-week-old chickens were inoculated with wild type CAV and viral load was determined in major target organs using qPCR. This study showed that exposure to CAV in older birds leads to similar levels of active viral replication to those seen in younger birds and results in subclinical disease. Finally, a study was carried out to determine whether VP2 alone can induce apoptosis. Site-directed mutagenesis was used to generate a point mutation that knocked out VP3 without imposing any change in the amino acid sequence of VP2. A pCAT-VP2+VP3- construct was generated and used to transfect MSB1 cells and the cells were examined using a TdT-mediated dUTP nick end labelling (TUNEL) assay to detect DNA strand breaks and 4'-6-diamidino-2-phenylindole (DAPI) staining to examine nuclear morphological changes. Analysis of TUNEL treated cells, which had also been stained with DAPI, showed an increased level of apoptotic death, indicating that VP2 can induce apoptosis in MSB1 cells. Further studies are needed to establish whether VP2 also has anti-apoptotic activity and to examine the kinetics of apoptotic and anti-apoptotic functions of this protein.